Project Summary/Abstract Magnesium is involved in most major metabolic processes, working as an enzymatic cofactor and playing a role in the stability of DNA and RNA. Despite this critical function, the mechanisms of magnesium homeostasis remain largely unknown. Much of our continued ignorance stems from the fact that, due to its critical role in the function of the cell and survival of the organism, evolution has ensured the rarity of disorders of magnesium homeostasis. Recently, an opportunity arose for elucidating the mystery of this basic biology in the discovery that patients with a rare defect in magnesium handling, known as Familial Hypomagnesemia with Secondary Hypocalcemia (HSH), were linked to mutations in the ion channel melastatin-related transient receptor potential 6 (TRPM6). In addition to being one of the few diseases of hypomagnesemia, HSH is one of few diseases linked to mutations in an ion channel. Furthermore, TRPM6 is exceptional among ion channels, as it is one of only two with an intrinsic kinase domain (the other being TRPM7). These unique features raise many questions, both of fundamental channel biology and the physiology of magnesium regulation. The current proposal is the first to scrutinize the dual function of the TRPM6 channel and kinase, investigating the mechanism by which TRPM6 mutations produce the HSH phenotype in patients. Our preliminary data suggests TRPM6 produces cleaved kinases (M6CKs) that interact with Mg2+ transporters and core members of the spliceosome assembly complex. This action is likely to be affected by TRPM6 channel conductance. We propose a mechanism whereby TRPM6 directs epithelial magnesium handling through localized ion signaling, triggering widespread kinase activity, and propose studies to delineate the contribution of the TRPM6 channel and M6CKs in this process. We will investigate the effect of TRPM6 conductance on the concentration of intracellular Zn2+, Mg2+, and Ca2+, as well as on the production of M6CKs. We will explore the effect of M6CKs on channel conductance and intracellular ion concentrations. These studies will shed light on the signaling advantages of coupling both channel and kinase in the same polypeptide. To define the functional role of M6CKs, we will investigate the action of M6CKs on target magnesium transporters and members of the spliceosome assembly complex. We will confirm our preliminary targets and conduct a transcriptome-profiling array to determine M6CKs' regulation of RNA splicing. Successful completion of this project is critical to defining the role of TRPM6 in mineral metabolism and will lay the foundation for future investigations in animal models. It is our hope that an improved molecular understanding of TRPM6 will identify new mechanisms maintaining mineral balance in humans and better define the pathophysiology of Familial HSH.